Summary

The three homeotic genes of the bithorax complex (BX-C), Ubx,
abd-A and Abd-B control the identity of the posterior thorax and
all abdominal segments. Large segment-specific cis-regulatory regions
control the expression of Ubx, abd-A or Abd-B in each of the
segments. These segment-specific cis-regulatory regions span the whole 300 kb
of the BX-C and are arranged on the chromosome in the same order as the
segments they specify. Experiments with lacZ reporter constructs
revealed the existence of several types of regulatory elements in each of the
cis-regulatory regions. These include initiation elements, maintenance
elements, cell type- or tissue-specific enhancers, chromatin insulators and
the promoter targeting sequence. In this paper, we extend the analysis of
regulatory elements within the BX-C by describing a series of internal
deficiencies that affect the Abd-B regulatory region. Many of the
elements uncovered by these deficiencies are further verified in transgenic
reporter assays. Our results highlight four key features of the iab-5,
iab-6 and iab-7 cis-regulatory region of Abd-B. First,
the whole Abd-B region is modular by nature and can be divided into
discrete functional domains. Second, each domain seems to control specifically
the level of Abd-B expression in only one parasegment. Third, each
domain is itself modular and made up of a similar set of definable regulatory
elements. And finally, the activity of each domain is absolutely dependent on
the presence of an initiator element.

INTRODUCTION

The three homeotic genes of the Drosophila bithorax complex
(BX-C), Ubx, abd-A and Abd-B, specify the identity of the
3rd thoracic segment (T3) and all abdominal segments (A1 to A8) of the fly
(Lewis, 1978;
Sanchez-Herrero et al., 1985).
These segments derive from parasegments (PS) 5 to 14 that form during early
embryogenesis. The expression of Ubx, abd-A and Abd-B is
regulated by a complex cis-regulatory region covering 300 kb of DNA. Molecular
genetic analysis has subdivided this large cis-regulatory region into nine
segment/parasegment-specific subregions [abd/bx, bxd/pbx, iab-2 to
iab-8 (Lewis, 1978;
Bender et al., 1983;
Karch et al., 1985;
Peifer et al., 1987;
Duncan, 1987;
Celniker et al., 1990;
Sanchez-Herrero, 1991) (for a
review, see Maeda and Karch,
2006)]. Remarkably, the genes and their regulatory regions are
aligned on the chromosome in the order of the body segments they specify.
Genetic studies using chromosomal rearrangement breaks have shown that when
cis-regulatory regions are removed from the complex, specific parasegments are
transformed into copies of more anterior parasegments. The clustering of
mutations with similar phenotypes along the chromosome has led to a model in
which each regulatory region is important in specifying homeotic gene
expression in one parasegment. For example, iab-5 is thought to
control the expression of Abd-B in PS10/A5, while iab-6 is
thought to control expression of Abd-B in PS11/A6.

Although it is clear that each specific cis-regulatory region is important
for the parasegment specific expression of a BX-C homeotic gene, it is still
unclear if each regulatory region works autonomously in this function.
Abd-B, for example, is expressed in PS10-13 in a stepwise gradient
from anterior to posterior. Its specific expression pattern is determined by
the four cis-regulatory regions iab-5 to iab-8. Because of
the stepwise increase in Abd-B expression and the fact that mutations
affecting the cis-regulatory regions transform parasegments into a copy of the
parasegment immediately anterior to them, it has been thought that the pattern
of Abd-B expression is the result of the additive effect of
sequential activation of the cis-regulatory domains
(Lewis, 1978;
Celniker et al., 1990).

How individual cis-regulatory domains work also remains a mystery. And
although many elements have been identified within the BX-C using transgenic
assays [such as initiation elements, maintenance elements (ME) cell type- or
tissue-specific enhancers, chromatin insulators and the Promoter Targeting
Sequence (PTS)], there is still a surprising lack of appropriate mutations to
determine how all of these elements interact and function within their native
environment (for a review, see Maeda and
Karch, 2006). Many of the mutations used to map the cis-regulatory
regions are chromosomal rearrangements breaks. Such breaks often separate more
than one regulatory region from its relevant target promoter, resulting in
homeotic transformations of multiple segments. Rearrangement breaks also
introduce foreign DNA next to the sequences of the BX-C, and so position
effects are observed. Because of these limitations, it has been very difficult
to confirm the function in the context of the BX-C of elements studied in
transgenes.

By mobilization of three P-element enhancer trap lines, we have isolated a
number of internal deficiencies that affect the iab-3 to
iab-8 cis-regulatory regions. Among the deficiencies isolated are
ones that remove initiator elements, ones that remove cell-type specific
enhancers, ones that remove insulators and two deficiencies that cleanly
removes two PTSs. Combining our genetic analysis with results from transgenic
reporter constructs allowed us to define two new initiators (IAB6 and IAB7a),
a new ME and various cell type-specific enhancers. Overall, our work provides
compelling functional evidence that the Abd-B region of the BX-C is
modular, with the expression of Abd-B in each parasegment being
primarily controlled by a single cis-regulatory domain. Our work also provides
strong genetic evidence for a temporal transition in cis-regulation, in which
the initiator elements dictate the later activity state for an entire
cis-regulatory domain.

MATERIALS AND METHODS

Plasmid constructions

Overlapping fragments from the walk
(Karch et al., 1985) have been
inserted upstream from the Ubx-lacZ reporter gene in the
CasperUbx-lacZ P-element vector
(Qian et al., 1991). All
cloning details are available upon request. Germline transformation was
performed essentially as described by Mihaly et al.
(Mihaly et al., 1997).
Positions of the constructs are given in nucleotide numbering along the BX-C
sequence of Martin et al. (Martin et al.,
1995). The coordinates of each of the fragments used for the
transgenic assays is listed in Table
1.

Names and positions of fragments inserted upstream of the
Ubx-lacZ reporter

Generating mutations

Internal deficiencies

The iab-5,6J81 and 5,6J82 deletions
were recovered during an experiment aimed at isolating local hops of the
bluetail transposon (Galloni et
al., 1993; Mihaly et al.,
1997). A chromosome (called J82.blt) containing the deletion
present in iab-5,6J82 and the starting bluetail transposon
was first isolated. In order to assign the phenotype associated with the
deletion alone, we remobilized the transposon from J82.blt Among the progeny,
we recovered iab-5,6J82 (a clean excision) and the
iab-5,6J81, which probably resulted from an aborted gene
conversion event. The iab-6,7IH, Fab-6,71 and
Fab-6,72 deletions were recovered by imprecise excision of
the bluetail transposon. Local hops and imprecise excision of the
iab-7blt transposon were screened on the basis of their
failure of complementation with the iab-4,5,6DB
chromosome. The extents of the deletions were mapped by whole-genome Southern
and by PCR analysis. Fab-3,5DVwas recovered on the basis
of its dominant phenotype during an excision screen of the HCJ200 P-element
present in iab-4 (Bender and
Hudson 2000). The iab-7280 deletion was
recovered by mobilization of the fs(3)5649 transposon, as described
by Barges et al. (Barges et al.,
2000).

PTSs deletions

iab-7R73

As meiotic recombination attempts to recover the R73 deletion separated
from the Fab-71 deletion had failed, we generated a new
R73 deletion by gene conversion using the fs(3)5649 chromosome
(Gloor et al. 1991).
Convertant chromosomes carrying the R73 deletion were screened by PCR. TheΔ
330 deletion was also recovered during the screen, probably resulting
from an abortive conversion event.

ΔHS*iab-6

Gene conversion initiated by the excision of the P-element in the
iab-7blt line was used to direct specific mutations in the
Fab-7 boundary (for details, see
Hogga et al., 2001). A
deletion removing the sequence covering the minor nuclease hypersensitive site
was created by first introducing two loxP sites in the NcoI and
BsmI sites at positions 84,827 and 85,028, respectively. The deletion
was recovered after a cross to flies expressing Cre
(Siegal and Hartl, 1996).

RESULTS

Isolation of internal BX-C deletions

In order to dissect genetically the 3′ cis-regulatory domain of the
Abd-B gene, we isolated a number of deficiencies derived from the imprecise
excisions of three P-elements [iab-7blt, fs(3)5649 and
HCJ200; see, respectively, Galloni et al.
(Galloni et al., 1993), Barges et al.
(Barges et al., 2000) and
Bender and Hudson (Bender and Hudson,
2000)]. Most of these deficiencies were recovered because of their
homeotic transformations of the posterior abdominal segments.

The deficiency breakpoints were first determined by whole genome Southern
analysis and then confirmed by PCR analysis. Their positions are reported
along the DNA sequence (SEQ89E) (Martin et
al., 1995). The homeotic transformations associated with these
deficiencies are described below, specifically focusing on the features of the
posterior abdominal segments in adult males. The Abd-B expression
pattern observed in the ventral nerve chord of mutant embryos is also
described. Fig. 1A shows the
extents of these deficiencies. The iab cis-regulatory domains are
positioned relative to the boundaries and data presented below.

Identification of the IAB6 and IAB7 initiators

Males homozygous for iab-7280 have their 7th abdominal
segment completely transformed into a copy of A6
(Fig. 2B). In mutant embryos,
the Abd-B expression pattern in PS12/A7 is replaced by the
PS11/A6-specific pattern (Fig.
3). The homeotic transformations and the Abd-B expression
patterns observed in iab-7280 are exactly the same as
those seen in iab-7Sz, a mutation that removes the entire
iab-7 cis-regulatory region between Fab-7 and Fab-8
(Galloni et al., 1993). In
agreement with this, the iab-7280 mutation deletes 20
kilobases (kb) between sequence positions 64,370 and 84,012
(Fig. 1A), thus removing the
entire iab-7 cis-regulatory region. On the proximal side, a second
smaller deletion occurred, removing DNA belonging to iab-6, between
89,392 and 86,650 (Fig. 1A) The
wild-type appearance of PS11/A6 in adults and embryos indicate that no
essential function of iab-6 is affected by this deletion.

The iab-6,7IH mutation deletes 26 kb (96,559-70,265;
Fig. 1A), removing about
three-quarters of the iab-6 region and nearly two-thirds of the
iab-7 region. In flies homozygous for this deletion, both A6 and A7
are transformed to differing degrees towards A5, consistent with the removal
of important iab-6 and iab-7 enhancers
(Fig. 2D). The adult phenotype
of this deletion shows that although iab-6 activity is completely
abolished (as seen by the complete transformation of A6 into A5),
iab-7 is still partially functional [revealed by the only partial
transformation of A7 into A5 (Fig
2D)]. The Abd-B expression pattern in the embryonic nerve
chord shows remarkable correspondence to these adult phenotypes
(Fig. 3). In PS11/A6, where
iab-6 is inactivated, Abd-B expression follows the
PS10/A5-specific pattern. If iab-7 were also completely inactive, we
would predict that the PS10/A5-specific Abd-B expression pattern
would also be reiterated in PS12/A7. Instead, we observe a weaker version of
the normal PS12/A7-specific pattern that corresponds to a level of expression
intermediate between that of normal PS11/A6 and PS12/A7.

Comparing iab-6,7IH to iab-7280
allows us to map important regulatory elements within the iab-6 and
iab-7 regions. The iab-7280 deletion does not
alter iab-6 function, while iab-6,7IH deletion is
null for iab-6. This indicates that sequences crucial for
iab-6 function lie between the proximal breakpoints of these two
deletions (89,392 and 96,559; see Fig.
1A). Meanwhile, regarding iab-7, the comparison of these
two deletions demonstrates that there are at least two separable elements
important for iab-7 activity. One element uncovered by the
iab-6,7IH deletion and one located in between the distal
breakpoints of the two deletions. Consistent with this, we and others had
previously isolated a weak PS12 specific initiator element mapping between the
distal breakpoints of these two deletions (67,072 and 67,806)
(Zhou et al., 1999;
Barges et al., 2000). We
postulate the existence of another PS12-specific initiator element.

Regulatory landscape of the Abd-B gene of the BX-C.
(A) Genomic map of the 3′ cis-regulatory region of the
Abd-B gene. The proximal and distal arrows point towards the
centromere and telomere, respectively. The black numbers above the DNA line
correspond to the nucleotide sequence marked in kb (SEQ89E)
(Martin et al., 1995). The
exons of the Abd-B transcription unit encoding the m variant are
shown below the DNA line. The extent of the iab-5, 6 and 7
cis-regulatory domains are indicated by brackets, with the domain boundaries
in between shown by gray circles. The red boxes on the DNA line mark the IAB5,
IAB6, IAB7a, IAB7b and IAB8 initiator elements (from proximal to distal,
respectively). The extent of the deletions analyzed are shown below the DNA
line, in black for deletions associated with loss-of-function of phenotypes
and in red for gain-of-function phenotypes. (B) The DNA line with the
scales as in A is drawn again. The extent of the different restriction
fragments used in transgenic constructs are shown below, with the initiator
fragments drawn in red. The expression patterns directed by these initiator
fragments are presented in C-F, which show embryos at the extended germ
band stage, doubled stained with antibodies directed against Engrailed (brown)
and the β-Galactosidase (in blue). The parasegmental anterior border of
lacZ expression (indicated by an arrow) shifts one parasegment
posterior in each panel from left to right

In order to further narrow down the regions containing these enhancers, we
cloned the regions implicated by our genetic analysis in front of a
Ubx-lacZ reporter gene and studied the expression pattern of the
resulting constructs in transgenic flies.

Genetic analysis had localized the new iab-6 regulatory element to
a ∼7 kb region between the proximal break points of the
iab-6,7IH and the iab-7280 deletions.
When a ∼8 kb fragment approximating this region is placed in front of the
Ubx-lacZ reporter construct (fragment 7.8H in
Fig. 1B), it drives expression
of the lacZ reporter in a parasegment-specific manner, with strong
expression in PS11, PS13 and PS14 (data not shown, but see below). The
parasegment-specific expression pattern driven by this element suggests that
it is capable of reading the parasegmental address and is, therefore, an
initiator element. By subcloning overlapping neighboring fragments into the
same vector, we were able to narrow down the iab-6 initiator to a∼
3 kb region contained within fragment 7.8H. Consistent with this, a∼
2.8 kb fragment (2.8XN in Fig.
1B) from this region is fully able to recapitulate the PS-specific
pattern of fragment 7.8H (Fig.
1D). We have therefore, named this fragment IAB6.

Homeotic transformations in adult male. Male abdomens were cut along
the dorsal midline and flattened on a slide. The dorsal surface of each
abdominal segment has a rectangular plate of hard cuticle called the tergite.
Only half of the tergites of the 4th, 5th and 6th abdominal segments
(numbered) are visible, as well as the genitalia at the bottom. (A) In
wild type, the 5th and 6th tergites are pigmented. The ventral surface of
abdominal segments is composed of soft cuticle called the pleura. On the
ventral midline of the pleura, there are small plates of harder cuticle called
sternites. In wild type, the 6th sternite (shown by arrows) can easily be
distinguished from the more anterior sternites by its different shape and by
the absence of bristles. In wild-type males, the 7th abdominal segment present
in embryos and larvae does not contribute to any adult structures after
metamorphosis. (B) iab-7280males harbor a complete
transformation of A7 into A6, as revealed by the presence of a large 7th
tergite and sternites (arrows). (C) In iab-7R73
males, however, there is only a weak transformation of A7 into A6, as revealed
by the small 7th tergite and the absence of a 7th sternite on the ventral
side. (D) In iab-6,7IH, A6 is completely
transformed into a copy of A5. However, A7 is only partially transformed into
a segment of intermediate identity between A5 and A6, as seen by the shape of
the 7th sternite, which resembles the 6th, but harbors a few bristles (A5
character). This indicates that in iab-6,7IH, iab-7 is not
completely inactivated (E)In iab-5,6J82, both A5
and A6 are transformed into a copy of A4. (F) In
iab-5,6J81 the residual pigmentation on the 6th tergite,
as well as the shape of the 6th sternite show that A6 is not fully transformed
into A4, but instead, into a mixture of identity between A4 and A6, suggesting
that the iab-6 domain is not completely inactivated by the deletion.
(G) In iab-4,5,6DB, A5 is transformed into A3.
However, as in iab-5,6J81, the 6th segment harbors
features of A6, suggesting that iab-6 is also not completely
inactivated by the deletion.

The IAB6 fragment is capable of maintaining strong lacZ expression
in PS11 and PS13 at later stages of embryogenesis (see Fig. S1B in the
supplementary material), indicating that the fragment contains a maintenance
element in addition to the initiator. The presence of a maintenance element is
further supported by the observation that transgenic flies carrying the IAB6
construct show pairing sensitive repression of the mini-white reporter gene in
30% of the lines (three out of 10 lines), a hallmark characteristic of MEs
(Kassis, 1994;
Gindhart and Kaufman, 1995;
Pirrotta and Rastelli,
1994).

To identify the second putative iab-7 activating element, we once
again cloned overlapping fragments from the implicated area into the
Ubx-lacZ reporter construct. A ∼3 kb fragment (3H;
Fig. 1B) located at the
proximal edge of the iab-7 cis-regulatory region is able to direct
the early expression of the Ubx-lacZ reporter gene in PS12
and PS14, indicating that this element is also an initiator element
(Fig. 1E). This expression
pattern is very similar to that already described for another PS12 initiator
element located about 10 kb away, at the other edge of the iab-7
cis-regulatory region (Zhou et al.,
1999; Barges et al.,
2000). We will refer to these two initiator elements as IAB7a and
IAB7b, with IAB7a being the more proximal element. It should be noted that the
level of PS12 and PS14-specific expression of IAB7a is much higher than that
of the previously described IAB7b element. In transgenic lines carrying IAB7a,
the lacZ expression pattern fades away at later stages of
embryogenesis, indicating that no maintenance element is present within this
fragment.

Abd-B expression pattern in wild type and deficiency
mutants. HRP staining with antibodies against Abd-B. After
staining, the central nervous systems were dissected out from 12 hour-old
embryos. In wild type, the typical Abd-B expression pattern is
characterized by an anterior-to-posterior gradient from PS10 to 13 in the
number of expressing nuclei per parasegment, as well as by the intensity in
each nucleus. See text for the description of the expression patterns in the
different mutants.

Initiators are not sufficient for Abd-B patterning

Homozygous iab-5,6J82 males have their A5 and A6
segments transformed into A4, indicating that both iab-5 and
iab-6 are affected by this mutation
(Fig. 2E). These adult homeotic
transformations are paralleled by the absence of Abd-B staining in
PS10/A5 and PS11/A6 in homozygous iab-5,6J82 embryos
(Fig. 3). In agreement with the
complete loss of iab-6 function, the entire iab-6 domain is
deleted. However, despite the fact that iab-5 function is inactive,
the deletion affects only one-third of the iab-5 region
(Fig. 1A; 83,791-106,497). This
observation indicates that sequences necessary for iab-5 activity
reside within the distal one-third of the iab-5 region. Consistent
with this finding, Busturia and Bienz
(Busturia and Bienz, 1993) had
previously identified an IAB5 initiator in this region that can drive
Ubx-lacZ expression in PS10, PS12 and PS14 (see also
Fig. 1C).

The iab-5,6J81 deletion shares its proximal endpoint
with iab-5,6J82, but it is much shorter and extends only
10.5 kb distally, removing the proximal one-third of iab-6
(Fig. 1A; between 95,869 and
106,497). The transformation of PS10/A5 into PS9/A4 and the loss of
Abd-B expression in PS10 corroborate that sequences covering the IAB5
initiator element are indispensable for iab-5 function
(Fig. 2F;
Fig. 3). Interestingly,
iab-6 function is partly affected in iab-5,6J81,
even though the IAB6 initiator is untouched by this deletion. In embryos, the
level of Abd-B expression appears normal in PS11/A6
(Fig. 3). However, in
homozygous adult males, there is a partial transformation of A6 into A5
(Fig. 2F). Although our
previous data indicates that the IAB6 initiator is required for iab-6
activity, we must conclude, based on iab-5,6J81, that it
is not sufficient for iab-6 function and that there are other
elements, probably adult tissue-specific enhancers that are required for full
iab-6 activity.

The iab-4,5,6DB deletion has already been described in
previous reports (Karch et al.,
1985; Celniker et al.,
1990; Sanchez-Herrero,
1991; Crosby et al.,
1993). It removes DNA between map positions 95,464 and 124,218
(Fig. 1A). Based on the
PS-specific expression pattern of enhancer trap P-elements that are inserted
in the BX-C (the HCJ200 line); Bender and Hudson
(Bender and Hudson, 2000) have
redefined the position of the iab-4 cis-regulatory region at position
127,367. Thus, iab-4,5,6DB appears to remove most of
iab-4, the entire iab-5 region and the proximal half of the
iab-6 cis-regulatory region (Fig.
1A). In agreement with this, homozygous
iab-4,5,6DB males have their 4th and 5th abdominal
segments transformed into the 3rd (Fig.
2G) (Karch et al.,
1985).

Interestingly, the 6th abdominal segment in homozygous
iab-4,5,6DB males is partly transformed towards A3 or A4
(Fig. 2G), indicating that the
remaining half of iab-6 is only partially functional even though the
IAB6 initiator is still present. The latter observation supports the
conclusions drawn from iab-5,6J81, which suggests that the
IAB6 initiator is necessary but not sufficient for iab-6 function. This
conclusion is further corroborated by the similarity of the embryonic
Abd-B expression pattern in PS10/A5 and PS11/A6 of embryos homozygous
for either iab-4,5,6DB or iab-5,6J81
(Fig. 3). In both cases, there
is no expression in PS10/A5 (both deletions remove iab-5), and
Abd-B expression in PS11/A6 is very similar to the wild-type
pattern.

Deletion of the iab-7 and iab-6 PTSs

We have isolated two deletions in the iab-7 region that are of
special interest as they remove the region implicated in Promoter Targeting
Sequence (PTS) activity. Zhou and Levine
(Zhou and Levine, 1999)
studied an element from the BX-C in reporter constructs. They suggested that
this PTS element helps a distal enhancer bypass an intervening insulator to
reach its target promoter. Zhou and Levine mapped the PTS to an 820 bp region
deleted from the Fab-7R73 chromosome. Unfortunately, this
chromosome was isolated as a revertant of Fab-71
(Gyurkovics et al., 1990) and
contains the Fab-71 deletion, making phenotypic analysis
difficult. We have recovered a chromosome carrying solely the 820 bp deletion
from the Fab-7R73 chromosome implicated in PTS activity,
that we named iab-7R73
(Fig. 1A; deletion of DNA
between 64,229 and 65,049; see Materials and methods).
Fig. 2C shows a homozygous
male. A5 and A6 acquire normal identity, while A7 is weakly transformed into
A6. Thus, a clean removal of the PTS sequence only weakly impairs
iab-7 function, with no effect on iab-5 and iab-6
function.

We also isolated a smaller deletion (Δ330iab7)
that shares the same proximal breakpoint with iab-7R73 but
extends only ∼670 bp (Fig.
1A). As homozygous Δ330iab7 males and
females are indistinguishable from wild-type flies, sequences crucial for the
iab-7R73 phenotype must lie within the 152 bp region on
the distal side of the iab-7R73 deletion.

Recently, a second PTS element originating from iab-6 has been
described (PTS-6) (Chen et al.,
2005). This second PTS element was mapped to the minor DNase I
hypersensitive site of Fab-7
(Karch et al., 1994) (from
positions 84,827 to 85,028). During the course of site-directed mutagenesis
studies on the Fab-7 boundary and the iab-6 cis-regulatory
domain, we isolated a ∼700 bp deletion (84,661-85,367) that removes the
Fab-7 minor hypersensitive site and ∼500 bp of iab-6
that we have named ΔHS*iab6. AlthoughΔ
HS*iab6 entirely removes the new PTS-6 element, it
is not associated with any abnormal phenotype.

Mutations associated with gain-of-function phenotype. (A-C)
Cuticles from adult males of homozygous Fab-3,5DV, wild
type and Fab-6,71. See text and the legend of
Fig. 2 for descriptions.
(D-F) The corresponding Abd-B expression patterns in the CNS
of 12-hour-old embryos (refer to legend of
Fig. 3).

Deletions associated with gain-of-function/boundary phenotypes

Fab-6,71 and Fab-6,72 are two
deletions associated with gain-of-function phenotypes with very similar
breakpoints and phenotypes. Both deletions share the same distal deficiency
breakpoint at position 83,791 kb (the site of insertion of the blt
transposon). On the proximal side, however, the deficiency breakpoint of
Fab-6,71 maps at position 101,096, while that of
Fab-6,72 maps to position 102,687
(Fig. 1A). Both of these
deletions remove the Fab-7 boundary element along with the entire
iab-6 domain. Interestingly, instead of showing a homeotic
transformation of the 6th abdominal segment into the 5th, like
iab-6,7IH, Fab-6,71 and
Fab-6,72 exhibit a striking dominant gain-of-function
phenotype, in which, the 5th and 6th male abdominal segments are largely
reduced in size (Fig. 4). In
extreme cases (such as shown in Fig.
4C), homozygous males harbor only rudiments of the 5th and 6th
abdominal segments. This phenotype corresponds to a strong transformation of
A5 and A6 towards A7 (in wild-type males, the 7th abdominal segment present in
embryos and larvae does not secrete any visible tergite or sternite cuticle).
In the CNS of embryos, we find the same level and pattern of Abd-B
expression in PS10/A5 and PS12/A7. These patterns are higher than the
wild-type PS10/A5 expression pattern and lower than the pattern in PS12/A11
(Fig. 4F).

Based on the similarity of these phenotypes to the previously isolated
Fab-7 and Fab-8 deletions, we believe that the
Fab-6,7 deletions must delete a putative Fab-6 boundary
element that normally separates iab-5 from iab-6. The
presence of this boundary element can be best observed by comparing the
Fab-6,7 mutations to the iab-6,7IH deletion.
Although in the Fab-6,7 mutants, we observe a fusion between
iab-5 and iab-7, in iab-6,7IH mutants,
iab-7 is still capable of behaving in an autonomous manner. These
observations imply that there must be a boundary element mapping to the region
between the proximal breakpoints of these deletions (96,559 and 101,096) that
keeps these domains separate.

Comparing the extent of the Fab-6,71 (or
Fab-6,72) mutation with that of the
iab-5,6J82 mutation sheds light on the dominant
gain-of-function frontabdominal phenotype. All three deletions share the same
distal endpoint, indicating that the origin of the
Fab-6,71 and Fab-6,72 GOF phenotypes
must lie within the DNA segment that remains present in
Fab-6,71 and Fab-6,72, and that is
deleted in iab-5,6J82. Interestingly, this DNA segment
contains the IAB5 initiator element (see above)
(Busturia and Bienz, 1993)
that, in the Fab-6,7 mutants, now becomes juxtaposed to the complete
iab-7 domain (the Fab-7 boundary is removed in the
Fab-6,7 alleles, and in iab-5,6J82). We believe
that the dominant gain-of-function phenotype is caused by the IAB5 initiator
element ectopically activating iab-7 in PS10, an interpretation
corroborated by the observation that in both mutations, the PS10/A5 and
PS12/A7 Abd-B expression levels are similar and higher than the level normally
found in PS10 of wild-type embryos.

Surprisingly, in both Fab-6,7 deletion mutants, PS11 expression of
Abd-B differs from that seen in PS10 and PS12. As the iab-6
domain is entirely deleted in these flies, we expected to see an A7/A5 fusion
pattern of expression similar to that seen in PS10 and PS12. Interestingly,
this is not the case. The pattern of Abd-B expression in PS11 is
greatly reduced from the level seen in its neighboring parasegments. As other
large boundary mutants, such as the Fab-3,5DV mutation
described below, do not behave similarly, we cannot explain this result at
this time.

The third deletion we identified that has a gain-of-function phenotype is
Fab-3,5DV. Fab-3,5DV is a deletion derivative
of the HCJ200 enhancer trap line that lies at the distal edge of
iab-3 at position 127,367 (Bender
and Hudson, 2000). An imprecise excision created a bidirectional
deletion of 25 kb between positions 112,381 and 137,391, removing the
Mcp boundary along with the entire iab-4 region and about 10
kb of the distal side of iab-3. Thus, Fab-3,5DV
fuses the proximal side of the iab-3 region to iab-5. The
deletion does not affect the previously identified IAB3 initiator element
(Simon et al., 1990). In
Fab-3,5DV, it appears that the juxtaposition of the IAB3
initiator element to iab-5 results in the ectopic activation of
iab-5 in A3/PS8. Adult males have A3 and A4 transformed into a copy
of A5 (Fig. 4A). Likewise, in
embryos, the Abd-B expression pattern normally seen in PS10 appears
ectopically in PS8 and PS9 (Fig.
4D).

Other regulatory elements in the Abd-B 3′ cis-regulatory
region

We have extended the reporter gene analysis that allowed us to identify the
IAB6 and IAB7a initiator elements, to scan the whole Abd-B 3′
cis-regulatory region for additional enhancers and maintenance elements. We
did this by inserting overlapping DNA fragments
(Fig. 1B) spanning most of the
region from iab-5 to iab-8 in front of the Ubx-lacZ
reporter gene in transgenic flies and studying either the expression pattern
of the resulting constructs, or the pairing-sensitive repression of the
mini-white cassette contained on the transgene (see Fig. S1 in the
supplementary material).

DISCUSSION

The proper identity of PS10/A5 through PS13/A8 is determined by the
anterior-to-posterior stepwise increase in the level of Abd-B. This
pattern of expression is achieved through the action of four large
cis-regulatory domains. Using reporter constructs, a number of groups have
identified several types of regulatory elements from within the cis-regulatory
region. These elements include initiator elements, maintenance elements
(polycomb response elements/PREs), cell/tissue-type specific enhancers,
chromatin insulators and promoter targeting sequences (PTSs) (for a review,
see Maeda and Karch, 2006).
Unfortunately, with the exception of the boundary elements, genetic proof for
the role of each of these elements has been confused because of the lack of
discrete mutations. Thus far, most of the mutations used for the genetic
analysis were rearrangement breaks (Lewis,
1978; Karch et al.,
1985; Sanchez-Herrero et al.,
1985; Celniker et al.,
1990). Because these breaks remove extremely large regions of the
cis-regulatory region and place foreign DNA in its place, the phenotypes
resulting from these mutants are often complex and difficult to interpret.
Here, we have described a collection of internal deletions within the
Abd-B region of the BX-C. Using these deletions, we have been able to
infer genetically the role of many of the elements previously identified. Our
results, when combined with previous data, point to four important conclusions
regarding the Abd-B region of the BX-C. First, as already discussed
in prior publications (Karch et al.,
1985; Celniker et al.,
1990; Sanchez-Herrero,
1991; Galloni et al.,
1993; McCall et al.,
1994; Bender and Hudson,
2000) (for reviews, see Peifer
et al., 1987; Maeda and Karch,
2006), the Abd-B region is modular by nature and can be
divided into discrete functional units or domains. Second, in a wild-type
situation, each domain appears to be sufficient to specify the appropriate
level of Abd-B expression in a particular parasegment. Third, each
domain is itself modular and made up of a similar set of definable regulatory
elements. Finally, the activity of each domain is absolutely dependent on the
presence of an initiator element.

Abd-B is controlled by a single iab domain in each
parasegment

In 1978, E. B. Lewis first proposed a model for BX-C patterning that stated
that once segment-specific functions are activated along the anteroposterior
axis, they remain active in more posterior segments and contribute to the
identity of those segments (Lewis,
1978). His hypothesis was based primarily on the posterior to
anterior transformations observed in flies lacking distal regions of the BX-C.
For example, embryos that lacked all BX-C sequences distal to the bxd
domain, had all abdominal segments posterior to the first abdominal segment
(A1) transformed into copies of A1. The transformations toward A1 suggested
that everything necessary for A1 development was present in all posterior
segments and that it was an accumulation of other segment-specific factors
that caused the deviation in segmental identity. In agreement with the Lewis
model, we, and others, have previously shown that enhancer trap lines inserted
in the BX-C and transgenic initiator constructs are expressed in a pattern
that starts from a specific parasegment and continues in more posterior
parasegments (Simon et al.,
1990; Qian et al.,
1991; Müller and Bienz,
1992; Galloni et al.,
1993; McCall et al.,
1994; Zhou et al.,
1999; Barges et al.,
2000; Bender and Hudson,
2000).

Although these findings strongly suggested that the domains that become
active in the parasegment they specify remain active in the more posterior
parasegments, our analysis of internal deletions in the BX-C indicate that
anterior domains are not required for the specification of more posterior
parasegments. This is best illustrated by deletions that remove most or all of
a single cis-regulatory domain. For example, the iab-7Sz
and iab-7280 deficiencies both delete the entire
iab-7 domain. If iab-7 was required for the identity of the
more posterior parasegment, PS13/A8, as predicted by the Lewis model, one
might expect that deleting iab-7 would affect PS13 identity. This is
not what is seen. Flies homozygous for either of these mutations have PS12/A7
transformed into PS11/A6, while PS11/A6 and, more importantly, PS13/A8 are not
affected in any detectable way. This phenotype indicates that although
iab-7 is absolutely required in PS12/A7, it is dispensable for the
identity of all other parasegments. However, PS12 is still transformed into a
perfect copy of PS11, indicating that iab-6 remains capable of
functioning in PS12/A7. Therefore, we believe that more anterior domains
remain capable of functioning in more posterior parasements, but only in the
absence of an active posterior domain. This is consistent, for example, with
the observation that bxd+ is required in segments more
posterior to PS6/A1, as bxd is the most posteriorly activated
regulatory domain of the Ubx gene.

Internal deletions that affect more than one cis-regulatory region confirm
these conclusions. For example, in iab-6,7IH, where both
the iab-6 and the iab-7 domains are deleted, both A6 and A7
acquire an A5 identity. However, PS13/A8 identity is again not affected by the
deletion even though, in this case, two more-anterior domains are missing.
Once again, the more-anterior iab-5 domain remains capable of acting
posterior to PS10/A5, but only in the absence of iab-6 and
iab-7. Similar results are seen with both the
iab-5,6J81 and the iab-5,6J82
deletions, where PS12/A7 identity is unaffected in the absence of
iab-5 (and also iab-6 for
iab-5,6J82).

Although it is possible that deletions of anterior domains lack a visible
phenotype because of the masking effect of the stronger enhancers in more
posterior domains, it is clear from our results that the graded Abd-B
expression pattern cannot be accounted for solely based on the summation of
individual regulatory domains. Instead, each regulatory domain is, by itself,
sufficient to generate an appropriate pattern of Abd-B expression in
a specific parasegment (see also Crosby et
al., 1993).

Initiators work as domain control regions

The autonomous nature of the cis-regulatory domains implies the existence
of elements within each domain that can independently sense and respond to
positional cues. At present, the best candidates for serving these functions
are among the enhancers identified by transgenic assays. Two basic types of
enhancers have been found within the cis-regulatory regions of the BX-C. Some
enhancers are capable of directing the expression of reporter genes in
specific types of cells or tissues, irrespective of parasegmental boundaries.
These enhancers are usually turned on relatively late during embryogenesis
(Simon et al., 1990;
Busturia and Bienz, 1993;
Pirrotta et al., 1995). An
example of this kind of enhancer is seen in fragment 4.4E from the
iab-6 cis-regulatory region that is capable of driving Abd-B
expression in the visceral mesoderm (see Fig. S1 in the supplementary
material). Other enhancers, however, direct expression of reporter genes from
the end of cellular blastoderm stage in a segmentally restricted manner,
indicating that they are capable of responding to a specific segmental address
(Simon et al., 1990;
Qian et al., 1991;
Müller and Bienz, 1992;
Shimell et al., 2000). These
include the previously identified IAB5, IAB7b and IAB8 enhancers that express
from PS10, PS12 and PS13, respectively
(Fig. 1C-F)
(Busturia and Bienz, 1993;
Zhou et al., 1999;
Barges et al., 2000). Here, we
described two new elements of this type: IAB6 with a PS11 specificity and
IAB7a with a PS12 specificity. Taken together, these findings demonstrate that
each cis-regulatory region contains at least one PS specific enhancer. These
specific enhancers have been previously referred to as `initiators'.

Analysis of the phenotype of internal deletions suggests that the removal
of the IAB5 or IAB6 initiator completely abolishes the function of
iab-5 or iab-6, respectively. For example, although the
iab-5,6J81 deletion leaves intact about two-thirds of the
iab-5 domain, it deletes the IAB5 initiator element and is completely
null for iab-5 function. The iab-6,7IH is another
example. This deletion removes the IAB6 initiator and, despite the fact that
one quarter of iab-6 is not affected by the deletion, iab-6
is completely inactivated. These results suggest that the IAB5 and IAB6
initiator elements are indispensable for iab-5 and iab-6
function, respectively.

Although the removal of initiator elements completely abolishes the
function of a regulatory domain, the presence of an initiator is not
sufficient to recapitulate the activity of the whole domain. This is clearly
seen in the iab-5,6J81 and iab-4,5,6DB
deletions. In both of these mutants, the IAB6 initiator is still present, yet
there is a partial transformation of A6 towards A5. These results highlight
the importance of other regulatory elements, such as cell-type-specific
enhancers, within the iab regions that help control Abd-B
expression.

Thus far, all available data suggest that iab-5 and iab-6
each harbor only one initiator element. The situation in iab-7 and
iab-8, however, appears more complex. In both cases, two initiator
elements have been identified in each domain. Comparison of the
iab-7280 and the iab-6,7IH phenotypes
suggests that within iab-7, the two initiators contribute to the
final Abd-B expression pattern. Although iab-7280
is completely null for iab-7, the iab-6,7IH
mutation leaves the IAB7b initiator intact and does not completely inactivate
iab-7. This is consistent with the idea that complete loss of
iab-7 function can be achieved by only the removal of both PS12
initiators in cis.

iab-8 may represent another example of a regulatory region
containing more than one initiator element. An IAB8 initiator has been
identified between the Fab-8 boundary and the 3′end of the
Abd-B transcription unit (Fig.
1) (Zhou et al.,
1999; Barges et al.,
2000). However, mutations that separate the IAB8 element from the
Abd-B transcription unit have no detectable effect on iab-8
function, suggesting the presence of a second element within iab-8
that can compensate for the loss of the IAB8 initiator. Indeed, Estrada et al.
(Estrada et al., 2002)
described the existence of a second iab-8-specific initiator element
upstream of the Abd-Bm promoter.

Within the BX-C, Abd-B expression is strictly restricted to
specific parasegments in all tissue. However, in transgenic constructs, only
initiator elements seem to sense a parasegmental address. Outside the complex,
the cell- and tissue-specific enhancers found within the BX-C do not. For
example, although an isolated fragment, 11X, from the iab-7 region
directs the expression of a Ubx-lacZ construct in the epidermis of
each parasegment (see Fig. S1 in the supplementary material), normal
Abd-B expression in the epidermis is restricted to the parasegments
posterior to PS12. The discrepancy between the transgenic and the native
expression of Abd-B implies that something within each domain must
act to control the activity state of the entire domain. Because the initiator
elements seem to be required for the function of each domain and can sense
parasegmental position, we believe that initiator elements act as domain
control regions that sense a parasegmental address and relay this information
to all other elements within the domain. Although the exact mechanism of this
transmission is unknown, it is clear that a part of the mechanism involves the
regulation of maintenance elements.

Here, we have described two new maintenance elements in iab-6. The
occurrence of multiple MEs/PREs in a single cis-regulatory region has been
documented before and may represent the normal situation, rather than the
exception (Chiang et al.,
1995). The iab-5 cis-regulatory region, for example, also
contains at least two MEs (Busturia and
Bienz, 1993; Busturia et al.,
1997; Müller et al.,
1999). Although there has been only one ME identified within each
of the iab-7 and iab-8 cis-regulatory regions
(Hagstrom et al., 1997;
Mihaly et al., 1997;
Barges et al., 2000), it is
likely that they too, contain more than one ME. Supporting this, a deletion of
the single known iab-7 ME (PRE) has very little consequence on the
phenotype of PS12/A7, suggesting a functional redundancy with another ME
within iab-7 (Mihaly et al.,
1997).

Like cell-type-specific enhancers, maintenance elements in transgenic
constructs do not seem to be parasegmentally regulated. Indeed, one of the
most common assays used to identify new maintenance elements requires them to
maintain a pattern of expression initiated by the bx initiator
(Müller and Bienz, 1991;
Simon et al., 1993;
Chan et al., 1994;
Fritsch et al., 1999;
Busturia et al., 2001).
Therefore, like that of cell-type specific enhancers, ME function seems to be
dictated by initiator elements.

Domain boundaries and PTS

Confirmation of the role of initiator elements in the control of both
cell-type specific enhancers and MEs, comes from the analysis of a specific
class of gain-of-function mutants. These mutations result from the simple
deletions of regions located between the cis-regulatory domains. As a result
of these deletions, neighboring cis-regulatory domains fuse into single units
of gene regulation. Thus, these regions have been called domain boundaries to
highlight their role in separating domains. An example of a boundary deletion
is the Fab-7 deletion (Gyurkovics
et al., 1990; Mihaly et al.,
1997). In the Fab-7 deletion, less than 800 bp are
deleted yet there is an almost complete transformation of PS11/A6 into
PS12/A7. Surprisingly, the cells not transformed towards a PS12-type fate do
not become PS11 like, but PS10 like. Normally in PS11/A6, iab-6 is
active while iab-7 is silent. In Fab-7 mutants, one gets a
fusion of the two domains. The result of this fusion is that there are two
different parasegment specific initiator elements in the same domain. We
believe that the resulting phenotypes stem from the competition between these
two initiator elements on the various enhancers and maintenance elements. In
most cells in PS11, the iab-6 initiator correctly reads the
parasegmental address as PS11 and instructs all enhancers and maintenance
elements (including the stronger iab-7/PS12 enhancers) to turn on
Abd-B expression. Because of the iab-7 enhancers, there is
an overexpression of Abd-B in PS11 to the level of PS12. Meanwhile,
in other cells, the iab-7 initiator(s) correctly reads the
parasegmental address as PS11, remains silent, along with other elements of
the domain (including the elements originating from iab-6).

Initiators control the activity of whole domains. Two adjacent
domains are drawn, as well as the target Abd-B gene further to the
right. The thick blue rectangles symbolize silencing by the Pc-G
complex(es). The example on the left shows an initiation domain, while the
example on the right shows a non-initiated domain.

Although the genetic evidence highlighting a role for the boundary regions
in Abd-B gene regulation is quite clear, the mechanism by which
boundaries function is still mysterious. In transgenic constructs, boundaries
act as insulator elements: elements capable of blocking enhancer-promoter
interactions when intercalated between them
(Hagstrom et al., 1996;
Zhou et al., 1996;
Zhou et al., 1999;
Barges et al., 2000). The role
of boundaries as elements capable of separating domain specific enhancers and
silencers may allude to the validity of this transgenic assay. However, it is
clear that within the BX-C, boundaries cannot solely act as insulator
elements, and that there must be some mechanism by which these insulators can
be bypassed when in the proper context.

An element deriving from the region next to the Fab-8 boundary has
been proposed to aid distal enhancers in bypassing intervening boundaries.
This element has been termed the promoter targeting sequence (PTS)
(Zhou and Levine, 1999). In
transgenic constructs, the PTS permits distal enhancers to overcome the
insulating effect of Fab-8. However, the in situ function of the PTS
is much less clear. Part of the reason for this is that some of the phenotypes
originally attributed to a PTS deletion, such as the partial transformation of
A5 towards A4, are, in fact, the consequence of the haplo-insufficiency of the
Abd-B deletion used by Zhou and Levine. Another factor complicating
the evaluation of the function of the PTS in the BX-C is that, until now, the
only available mutation that deletes the PTS region was a revertant of
Fab-71 (Fab-7R73), and therefore
carried a second deletion in the Fab-7 region.

Here, we described two mutations that remove putative PTS elements. The
first is a mutation that deletes the R73/PTS-7 region without affecting the
rest of the BX-C. And the second is a deletion that removes the newly
described PTS-6 element from iab-6 (Chen
et al., 2005). In both cases, the phenotypes associated with the
PTS deletions are relatively small. In contrast to the phenotype of the double
deletion, Fab-7R73, flies homozygous for the single
iab-7R73 deletion exhibit only a rather mild
transformation of A7. The morphology of A6, however, is wild type in these
flies, indicating that the R73 region has only a weak regulatory activity that
is restricted to iab-7. Meanwhile, theΔ
HS*iab6 deletion that removes the entire PTS-6
element is completely wild type in appearance. Taken together, our results
indicate that if the PTS hypothesis is correct, each domain must contain
multiple, redundant PTS elements.

The domain model

Based on our data and the data of others
(Simon et al., 1993;
Crosby et al., 1993;
Pirrotta et al., 1995), we
propose a model to summarize our current understanding of how the BX-C
functions (Fig. 5). We believe
that each domain autonomously controls the expression of Abd-B within
one parasegment. Accordingly, each regulatory domain in the BX-C is a modular
array of all of the elements necessary for Abd-B expression in a
particular parasegment. Key to the functioning of each domain is the initiator
element. Acting as a centralized domain control region, the initiator element
determines whether the appropriate gap and pair-rule gene products are present
to justify the activation of the domain. If the proper positional address is
read, the initiator then sends a signal to the rest of the domain to activate
various enhancers. Because a domain lacking an initiator seems to be silenced,
we imagine that this signal is made up primarily of an inhibitory signal to
the ME/PREs. With the PREs placed into a non-silencing state, the enhancers
become free to activate Abd-B. If the proper parasegmental address is
not found, then the Pc-G proteins silence the domain. Throughout all of this,
the domain boundaries keep each domain independent and free from competing
influences.

Despite the fact that a rough map of the `regulatory landscape' of the
Abd-B domain is now available, a number of questions regarding the
function of the regulatory elements remain to be answered. These include the
problem of how different regulatory elements within individual domains
interact and communicate. For example, what is the nature of the signal from
initiator to ME? One possibility currently being discussed is the idea that
transcription across PREs might be the inhibitory signal
(Bender and Fitzgerald, 2002;
Hogga and Karch, 2002;
Rank et al., 2002;
Schmitt et al., 2005).
Although it is still unclear whether transcription plays an instructive role
in BX-C regulation, it is interesting to speculate that initiators may in fact
generate transcription across PREs to inactivate them. Until recently,
answering these types of questions within the BX-C have been unreasonable.
Perhaps now, through the use of targeted mutagenesis and gene conversion, we
will finally be able to tackle precise questions in the BX-C. Indeed, our
results raised many questions, now ripe for investigation.

Supplementary material

Acknowledgments

We thank Kirsten Hagstrom and Paul Schedl for the identification of the
iab-6PRE. We also thank Eva Favre and Jorge Faustino for excellent technical
assistance. This work was supported by the Swiss National Foundation, by the
Human Frontier Science Organization and by the Sate of Geneva. J.M. is an
EMBO/HHMI Scientist and a Bolyai János Research Scholar. H.G. is
supported by grants from OTKA and by the NIH as a subcontractor.